PROJECT SUMMARY Individuals that binge drink alcohol during adolescence are more likely to develop chronic, relapsing alcohol disorders at some point in their life. Studies in animals have also shown that repeated episodes of adolescent alcohol exposure lead to dysregulation of the medial prefrontal cortex (PFC) and deficits in inhibitory control and behavioral flexibility in adulthood. The goal of this proposal is to utilize a rodent model of adolescent intermittent ethanol (AIE) exposure to investigate mechanisms involved in alcohol-induced deficits in prefrontal function and behavioral control in adulthood. Recent research suggests that a subset of ventral tegmental area (VTA) dopaminergic projection neurons that initially innervate the nucleus accumbens (NAc) subsequently grow to the PFC, a process that is important for executive function in adulthood. While our lab has shown that AIE significantly impacts dopaminergic neurotransmission and regulation in the prelimbic (PrL) subregion of the PFC, it is unknown if the newly identified VTA-NAc-PrL pathway is altered by AIE exposure. Synaptic pruning in the medial PFC and NAc during adolescence is critical for inhibitory control and behavioral flexibility. Interestingly, accumulating data indicates that this process is facilitated through glial-mediated phagocytosis. Although it is known that alcohol can alter neuroimmune signaling, whether AIE exposure affects glial/neural interactions and glial morphology is not well understood. The overarching hypothesis of the current proposal is that AIE exposure reduces VTA innervation and neural/glial interactions in the PrL cortex, contributing to alterations in adult behavior. Specific Aim 1 will test the hypothesis that AIE exposure reduces the developmental innervation and synaptic refinement of VTA originating DA projections in the PrL cortex. This aim will use axon-initiated viral transduction, immunofluorescence, and advanced super-resolution confocal microscopy and 3D analysis to examine innervation of the PrL by VTA-DA neurons whose axons initially terminated in the NAc and then grew to the PrL during adolescence. In order to gain a broader perspective of alterations within the PrL-NAc circuit following AIE, studies will also examine glial interactions with PrL neurons that project back to the NAc. Specific Aim 2 will test the hypothesis that AIE and inhibition of the VTA-NAc-PrL projection will similarly alter active and passive avoidance in adulthood. This aim will gain insight into the impact of AIE and the VTA-NAc-PrL projection on behavioral flexibility. The novel platform-mediated avoidance task allows for the simultaneous investigation of both passive and active avoidance, an area in the developmental literature on AIE that has been relatively overlooked. Studies will also determine whether DREADD-mediated inhibition of the VTA-NAc-PrL pathway alters adult avoidance behavior in a similar manner to AIE. The novel conceptual nature of this proposal, in combination with state-of-the-art techniques and outstanding training resources will provide the PI with an increased skill set that will further the advancement towards a future as an independent academic researcher.